Qianben Wang, Ph.D. - US grants

DESCRIPTION (provided by applicant): The androgen receptor (AR), a ligand-dependent transcription factor, plays a key role in the onset and progression of prostate cancer and is a therapeutic target. Surprisingly little is known of AR binding, AR collaborating transcription factors, and regulation of AR target genes in the human genome. The overall goal of this proposal is to investigate the combinatorial transcriptional regulation of protein-coding genes and a class of non-coding genes (microRNA [miRNA]) by AR and its collaborating transcription factors from a genome-wide view in androgen-dependent (AD) and -independent (Al) prostate cancer cells. To address these issues, we will use chromatin immunoprecipitation (ChIP) combined with human whole genome interrogating tiling microarrays (ChlP-on-chip) to study in vivo binding of transcription factors and their regulatory function in AD and Al prostate cancer. Our specific aims are to: (1) Determine whether distinct AR binding, AR collaborating transcription factor partners and AR target genes exist in AD and Al prostate cancer cells. AR ChlP-on-chip assays will be performed in AD and Al prostate cancer cells. AR binding, its collaborating transcription factors and AR target genes will be predicted by bioinformatics algorithms and experimentally validated. (2) Determine how AR and its collaborating transcription factors combinatorially regulate AR target genes in AD and Al prostate cancer cells. Collaborating transcription factors ChlP-on-chip will be performed and correlated with AR ChlP-on-chip and gene expression profiles to identify combinatorial transcriptional regulatory codes for AR target genes in AD and Al prostate cancer cells. (3) Determine whether AR and its collaborating transcription factors regulate miRNAs in AD versus Al prostate cancer cells. RNA polymerase II (pol II) ChlP-on-chip will be performed in AD and Al prostate cancer cells. Pol II binding will be correlated to AR and its collaborating transcription factors bindings and miRNA expression profiles to identify differential transcription factors-regulated miRNA expression in AD and Al prostate cancer cells. In summary, these studies will increase our fundamental understanding of differential transcriptional regulation of target coding and non-coding genes by AR and its collaborating transcription factors on a genome-wide level in AD and Al prostate cancer, which will lead to identification of new molecular targets for therapeutic intervention in AD and Al prostate cancer.

DESCRIPTION (provided by applicant): Regulation of androgen receptor function by H3K4 methylation in prostate cancer Project Summary The evolution of prostate cancer from an androgen-dependent state (ADPC) to one that is castration- resistant (CRPC) marks the lethal progression of the disease. Understanding the pathogenesis of CRPC and development of novel therapies for CRPC remains an urgent need. The androgen receptor (AR), a ligand-dependent transcription factor, is still expressed and functional in CRPC;however, how AR regulates target genes in CRPC and the functional roles of AR target genes in CRPC is poorly understood. In preliminary studies we have found that AR selectively binds to enhancer regions of M- phase cell cycle genes (e.g. UBE2C) in a CRPC cell model but not in an ADPC cell model, leading to higher M-phase gene expression and faster growth of CRPC than of ADPC. Interestingly, we further found that increased histone H3 lysine 4 (H3K4) methylation level on the M-phase gene enhancers is the underlying mechanism for selective AR binding at M-phase gene enhancers in CRPC compared with ADPC. However, these studies are limited to identifying and characterizing a few enhancer H3K4 methyaltion regulated AR target genes in a pair of CRPC/ADPC cell models. In this proposal we hypothesize that enhancer and promoter H3K4 methylation directs AR in the global regulation of target genes involved in critical processes such as growth and invasion in CRPC. Our specific aims are to: (1) To determine whether the enhancer H3K4 methylation and AR play a causal role in regulating UBE2C expression and to investigate the functional role of UBE2C in various CRPC cell models and in tumorigenesis in vivo. The hypothesis that UBE2C is a direct enhancer H3K4 methylation and AR co- regulated gene that plays a critical role in growth and invasion of at least a subset of CRPC cell models and in tumorigenesis in vivo will be tested in this aim. (2) To globally identify and characterize enhancer/promoter H3K4 methylation and AR co-regulated genes in CRPC cells. The hypothesis that gain of H3K4me2 directs distal enhancer-bound AR to activate oncogenes, whereas loss of H3K4me2 and/or H3K4me3 leads to enhancer- and/or promoter-bound AR-mediated silencing of tumor suppressor genes in CRPC will be tested in this aim. (3) To examine the relevance of H3K4 methylation/AR regulation of target genes in human prostate cancer samples. The hypothesis that the data obtained from Aims 1 and 2 is relevant to human prostate cancer will be evaluated in CRPC and ADPC samples in this aim. PUBLIC HEALTH RELEVANCE: Regulation of androgen receptor function by H3K4 methylation in prostate cancer Relevance Altered histone H3K4 methylation in castration-resistant prostate cancer may direct androgen receptor in the regulation of downstream target genes involved in critical processes such as growth and invasion. This proposal will significantly increase our understanding of the molecular and cellular outcomes of H3K4 methylation and AR co-regulation in prostate cancer, which has translational implications in the development of new therapies and identification of new biomarkers in prostate cancer.

ABSTRACT/SUMMARY ? Overall Systems Analysis of Epigenomic Architecture in Cancer Progression Despite anti-hormone therapies in patients, the cognate receptors ER? and AR can remain functional to support oncogenic signaling for advanced progression of breast and prostate cancers. Intensive studies have uncovered cellular and biochemical changes underlying the development of hormone resistance. However, epigenetic mechanisms for establishing and maintaining a hormone-resistant phenotype remain to be explored. Our preliminary studies have found remarkably similar epigenetic machineries that regulate hormone-independent gene transcription in both breast and prostate cancers. This process has multifaceted components, involving trans- and cis-acting elements, nucleosome reorganization, and chromatin interactions. To understand this complex mechanism, the San Antonio-Ohio State University Research Center for Cancer Systems Biology (SA-OSU RCCSB) has assembled a team of 21 experimental and computational investigators, and oncologists who will study a three-tiered epigenetic framework for gene regulation. First, microenvironmental cues initiate the recruitment of a specific combination of trans-bound transcription factors (TFs), called MegaTrans TFs, to ER? or AR-bound enhancers (Project 1). MegaTrans TFs are composed of diverse signaling-dependent transcription factors that activate these enhancers through receiving other signal cues without hormone stimulation. Second, this hormone-independent action requires well-orchestrated repositioning of nucleosomes, enabling maximal MegaTrans-DNA contact in target chromatin regions (Project 2). Pioneer factor FOXA1 and chromatin remodelers are also critical regulators of repositioned nucleosomes during the transition of a hormone-sensitive to -resistant phenotype. Third, this concerted action triggers chromatin movement, remotely bringing the MegaTrans/enhancer complexes in close proximity to target promoters (Project 3). Intra- and inter-chromatin interactions facilitate the formation of transcriptional architectures that efficiently and autonomously regulate ER?/AR-mediated gene expression even in the absence of agonists or in the presence of antagonists. Experimental investigators will use omics-seq platforms to map combinatorial MegaTrans complexes, repositioned nucleosomes, and topologically associated domains (TADs) that spatiotemporally regulate hormone-independent transcription. Computational scientists will then use omics data to derive 3D models of DNA-eRNA-protein interacting units in subnuclear compartments of cancer cells. Back to the bench, experimental scientists will use in silico findings to validate enhancer/gene markers that predict a hormone-resistant phenotype in patient-derived xenografts (PDXs) and clinical samples. To ensure seamless data integration of the three proposed projects, a Data Analysis and Management Core will implement customized toolkits to manage computational infrastructure and store omics-seq metadata for heuristic queries by community systems biologists. An Outreach Core will facilitate training of new-generation systems biologists and enhance collaborative efforts within the NCI's consortium and in the 4D nucleome community. An Administrative Core will provide governance and oversee rigorous evaluations of Intra-center Pilot Projects (IPPs), ensure cross-pollination between bench and in silico scientists in the SA-OSU RCCSB, and reinforce national guidelines of data sharing.

Project Summary/Abstract The evolution of prostate cancer from an androgen-dependent state (ADPC) to castration-resistant adenocarcinoma (CRPC) marks the lethal progression of the disease. Contemporary therapy for CRPC employs inhibitors of intra-tumoral and adrenal androgen synthesis (e.g. abiraterone acetate) or more potent androgen receptor (AR) antagonists (e.g. enzalutamide). However, these agents only provide a temporary response and modest increase in survival indicating a rapid evolution of resistance. In addition to CRPC, a significant number of patients develop small cell neuroendocrine carcinoma (SCNC) after hormonal therapy. SCNC is extremely aggressive and rapidly fatal. Importantly, with the widespread use of abiraterone acetate and enzalutamide, a greater frequency of the SCNC has been observed. Currently, there is no standard therapy that is effective for SCNC. Thus understanding the pathogenesis of CRPC/SCNC evolution and development of novel targeted therapies remain urgent needs. In addition, predictive and prognostic biomarkers that can help ?personalize? therapy and improve the precision of clinical trials are necessary. In preliminary studies, we have found that AKT-induced aberrant phosphorylation of transcription coactivator Mediator 1 (MED1) is required for UBE2C oncogene expression and for growth of CRPC and/or SCNC cells in vitro and in vivo. Furthermore, pharmacological AKT inhibition decreases UBE2C oncogene expression and cell growth in vitro in a phosphorylated MED1 (p-MED1)-specific manner. Importantly, the expression of p- MED1 significantly increases when human prostate cancer progresses to CRPC and SCNC. These findings support our hypothesis that AKT-induced aberrant MED1 phosphorylation drives an oncogenic gene expression program for CRPC/SCNC growth and progression. Our specific aims are to: (1) delineate the genomic mechanisms of p-MED1 binding to chromatin and global gene regulation in CRPC/SCNC; and (2) determine the biological impact and clinical relevance of AKT-p-MED1 transcriptional regulation in CRPC/SCNC. The successful completion of these aims will significantly impact the understanding of the critical oncogenic role of p-MED1 in CRPC and SCNC, laying the foundation for: (a) developing new therapies targeting the AKT-p-MED1 transcription axis, (b) employing p-MED1 as a marker for selecting AKT inhibitor- sensitive patients for ?precision medicine? clinical trials, and (c) identifying new predictive and prognostic biomarkers for CRPC and SCNC.

Novel genomic mechanism for ligand-dependent transcription by androgen receptor Project Summary/Abstract Androgen receptor (AR) is a member of nuclear hormone receptor (NR) superfamily that binds to cognate hormone responsive elements (HREs) and regulates target gene expression in an endogenous ligand (agonist)-inducible manner in diverse tissues. As AR plays a key role in the onset and progression of prostate cancer, numerous synthetic AR antagonists have been developed to inhibit the action of endogenous AR ligands. A prominent example is enzalutamide (Xtandi®), a second-generation AR antagonist showing strong anti-cancer activity for prostate cancer. However, intrinsic or acquired resistance to enzalutamide, and all available AR antagonists, occurs leading to treatment failure. Thus, therapeutic efficacy of current AR antagonists needs to be improved. Elucidation of genomic mechanisms underlying antagonist-liganded AR function is critically important in order to improve AR-targeted therapy. In preliminary studies, we have defined the first high-resolution (motif-resolution) agonist- and antagonist-liganded AR cistromes in prostate cancer cells by using a novel chromatin immunoprecipitation-exonuclease (ChIP-exo) approach. Unexpectedly, we found that AR bound to natural agonist (dihydrotestosterone, DHT) and antagonist (enzalutamide) recognizes distinctly different DNA motifs on chromatin (termed ?DNA motif switching?). Surprisingly, integrated ChIP-exo and RNA-seq analysis found that enzalutamide-liganded AR, bound to a novel AR binding motif, significantly affects global, cancer-relevant transcription. By combining our novel ChIP-exo genomic approach with other epigenomic, proteomic and biochemical approaches, we further found that enzalutamide-liganded AR interacts with specific collaborating transcription factors (e.g. FoxA1) and non-DNA binding coregulators (e.g. Hsp90) on specific active cis-regulatory regions. Importantly, pharmacological Hsp90 inhibition significantly decreases expression of enzalutamide-liganded AR target genes (e.g. cancer promoting genes GR and CD55) and enhances cell growth inhibitory effect of enzalutamide. Based on these compelling data, we hypothesize that DNA motif switching is a novel genomic mechanism underlying antagonist-dependent, cancer-relevant transcription by antagonist-liganded AR transcription complex. Our specific aims are to: 1) determine whether specific transcription factors and epigenetic features globally facilitate AR DNA motif switching; and 2) investigate how antagonist-liganded AR binding regulates expression of cancer-relevant genes. By significantly enhancing our understanding of how antagonist-regulated transcription by AR is controlled at the genomic level, this study will lay the foundation for future development of improved AR-targeted therapy.

ABSTRACT/SUMMARY - Project 2 Fine-scale nucleosome repositioning of enhancers for hormone-independent genomic function The cognate receptors AR and ER? can remain active for tumor progression after anti-hormone treatment for patients with prostate and breast cancers. Despite intensive efforts to elucidate the underlying mechanisms, little information is available concerning AR/ER? genomic function for promoting hormone resistance at the nucleosome level. In preliminary studies, we observed this genomic function is well orchestrated, relying on precise nucleosome organization within cis-bound enhancers for hormone-dependent transcription. Interestingly, we also found that this epigenetic mechanism can be hijacked by hormone-resistant cells to gain their growth and invasion advantages. Therefore, we hypothesize that altered nucleosome positions, or nucleosome repositioning, in and near AR/ER?-bound enhancers is being exploited for hormone-independent genomic function in advanced cancers. In Aim 1, we will conduct ChIP-ePENS and MNase-seq to comprehensively map nucleosome boundaries of AR/ER?-bound enhancers in a panel of hormone-sensitive and -resistant cancer cells. RNA-seq will be conducted to determine differential expression patterns of corresponding genes in these cell lines. The NucPat computational pipeline will be deployed to seamlessly process complex omics-seq data (Aim 2). We will use a Kernel Density Estimation algorithm to determine nucleosome positioning and spacing when AR or ER? establishes direct contact with its binding motif. Using a Hidden Markov model, we will identify active nucleosome states that maximize DNA-protein contact for AR/ER? genomic functions. In addition, pioneer factor FOXA1 and chromatin remodelers participate in this nucleosome repositioning even in the absence of agonists or in the presence of antagonists. To confirm this computational modeling in vivo, ChIP-ePENS and MNase-seq will be conducted in patient-derived xenograft (PDX) lines carrying hormone-sensitive and -resistant tumors (Aim 3). A nucleosome-phasing index (NPI) will be established to quantitatively assess the nucleosome states of AR/ER? redeployment in different PDX lines. This integrative omics analysis will be extended to a cohort of primary tumors, categorized into high- and low-risk groups. Again, we will calculate individual NPIs and correlate the data with clinicopathological features of patients. This translational study is intended to determine whether nucleosome phasing for AR/ER? redeployment is already present in high-risk primary tumors. Patients with this intrinsic phenotype are expected to have an adverse clinical outcome, irrespective of their anti-hormone treatments. Therefore, our proposed study will address a previously uncharacterized mechanism of hormone resistance and provide experimental evidence that nucleosome repositioning plays an integral role in redefining AR/ER? genomic function for advanced development of prostate and breast cancers.